Methods for isolating tissue factor inhibitor

ABSTRACT

A cDNA clone having a base sequence for human tissue factor inhibitor (TFI) has been developed and characterized and the amino acid sequence of the TFI has been determined.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a division of U.S. Ser. No. 10/377,817 filed Mar. 4,2003, which is a continuation of U.S. Ser. No. 09/627,676 filed Jul. 28,2000, which is a continuation of Ser. No. 09/054,782 filed Apr. 3, 1998,now issued as U.S. Pat. No. 6,171,587, which is a continuation of Ser.No. 08/463,323 filed Jun. 5, 1995, now issued as U.S. Pat. No.5,849,875, which is a continuation of Ser. No. 08/355,351 filed Dec. 13,1994, now abandoned, which is a continuation of Ser. No. 08/093,285filed Jul. 15, 1993, now issued as U.S. Pat. No. 5,466,783, which is acontinuation of Ser. No. 07/566,280 filed Aug. 13, 1990, now abandoned,which is a division of Ser. No. 07/123,753, filed Nov. 23, 1987, nowissued as U.S. Pat. No. 4,966,852, which is a continuation-in-part ofapplication Ser. No. 07/077,366, filed Jul. 23, 1987, now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to a coagulation inhibitor known as tissue factorinhibitor (TFI) and alternatively as lipoprotein associated coagulationinhibitor (LACI). More particularly, the invention relates to a cDNAclone representing essentially the full size TFI.

The coagulation cascade that occurs in mammalian blood comprises twodistinct systems—the so-called intrinsic and extrinsic systems. Thelatter system is activated by exposure of blood to tissue thromboplastin(Factor III), hereinafter referred to as tissue factor (TF). Tissuefactor is a lipoprotein that arises in the plasma membrane of many celltypes and in which the brain and lung are particularly rich. Upon cominginto contact with TF, plasma Factor VII or its activated form, FactorVII_(a), forms a calcium-dependent complex with TF and thenproteolytically activates Factor X to Factor X_(a), and Factor IX toFactor IX_(a).

Early studies concerning the regulation of TF-initiated coagulationshowed that incubation of TF (in crude tissue thromboplastinpreparations) with serum inhibited its activity in vitro and preventedits lethal effect when it was infused into mice. Extensive studies byHjort, Scand. J. Clin. Lab. Invest. 9, Suppl. 27, 76–97 (1957),confirmed and extended previous work in the area, and led to theconclusion that an inhibitory moiety in serum recognized the FactorVII-TF complex. Consistent with this hypothesis are the facts that theinhibition of TF that occurs in plasma requires the presence of Ca²⁺(which is also necessary for the binding of Factor VII/VII_(a) to TF)and that inhibition can be prevented and/or reversed by chelation ofdivalent cations with EDTA. More recent investigations have shown thatnot only Factor VII_(a) but also catalytically active Factor X_(a) andan additional factor are required for the generation of TF inhibition inplasma or serum. See Broze and Miletich, Blood 69, 150–155 (1987), andSanders et al., Ibid., 66, 204–212 (1985). This additional factor,defined herein as tissue factor inhibitor (TFI), and alternatively aslipoprotein associated coagulation inhibitor (LACI), is present inbarium-absorbed plasma and appears to be associated with lipoproteins,since TFI functional activity segregates with the lipoprotein fractionthat floats when serum is centrifuged at a density of 1.21 g/cm³.According to Broze and Miletich, supra, and Proc. Natl. Acad. Sci. USA84, 1886–1890 (1987), HepG2 cells (a human hepatoma cell line) secretean inhibitory moiety with the same characteristics as the TFI present inplasma.

In copending application Ser. No. 77,366, filed Jul. 23, 1987, apurified tissue factor inhibitor (TFI) is disclosed which was secretedfrom HepG2 cells. It was found to exist in two forms, a TFI₁, migratingat about 37–40,000 daltons and a TFI₂ at about 25–26,000 daltons, asdetermined by sodium dodecylsulfate polyacrylamide gel electrophoresis(SDS-PAGE). A partial N-terminal amino acid sequence for the TFI wasassigned as:

1 X-X-Glu-Glu-Asp-Glu-Glu-His-Thr-Ile-Ile-Thr-Asp- 15  Thr-Glu-16                                             27Leu-Pro-Pro-Leu-Lys-Leu-Het-His-Ser-Phe-(Phe)-Alawherein X-X had not been determined. The disclosure of said applicationis incorporated herein by reference.

BRIEF DESCRIPTION OF THE INVENTION

In accordance with the present invention, the complete coding sequenceof a cDNA clone representing essentially the full size tissue factorinhibitor (TFI) has been developed.

Initially, human placental and fetal liver λgt11 cDNA libraries werescreened with a rabbit polyclonal antiserum raised against a purifiedTFI. Immunologically positive clones were further screened for¹²⁵I-Factor X_(a) binding activity. Seven clones were obtained whichwere immunologically and functionally active. The longest clone,placental-derived λP9, was 1.4 kilobases (kb) long while the other sixwere 1.0 kb in length. Partial DNA sequencing showed the 1.0 kb clonesto have sequences identical to part of the longer 1.4 kb clone.Nucleotide sequence analysis showed that λP9 consisted of a 1432basepair (bp) cDNA insert that includes a 5′-noncoding region of 133 bp,an open reading frame of 912 bp, a stop codon, and a 3′-noncoding regionof 384 bp.

The cDNA sequence encodes a 31,950 Dalton protein of 276 amino acidswhich includes 18 cysteines and 7 methionines. The translated amino acidsequence shows that a signal peptide of about 28 amino acids precedesthe mature TFI protein. It will be understood that the “mature” TFI isdefined to include both TFI and methionyl TFI by virtue of the ATGtranslational codon in the λP9 clone described herein.

There are three potential N-linked glycosylation sites in the TFIprotein with the sequence Asn-X-Ser/Thr, wherein X can be any of thecommon 20 amino acids. These sites are at amino acid positions Asn 145,Asn 195, and Asn 256, when the first methionine after the 5′-noncodingregion is assigned amino acid position +1.

The translated amino acid sequence of TFI shows several discernibledomains, including a highly negatively charged N-terminal, a highlypositively charged carboxy-terminal, and an intervening portionconsisting of 3 homologous domains with sequences typical of Kunitz-typeenzyme inhibitors. Based on a homology study, TFI appears to be a memberof the basic protease inhibitor gene superfamily.

The original source of the protein material for developing the cDNAclone λP9 was human placental tissue. Such tissue is widely availableafter delivery by conventional surgical procedures. The λgt11 (lac5 nin5c1857 S100) used herein is a well-known and commonly available lambdaphage expression vector. Its construction and restriction endonucleasemap is described by Young and Davis, Proc. Natl. Acad. Sci. USA 80,1194–1198 (1983).

Northern blot analysis showed that the following liver-derived celllines: Chang liver, HepG2 hepatoma, and SK-HEP-1 hepatoma, all contained2 major species of mRNA (1.4 and 4.4 kb) which hybridized with the TFIcDNA.

The cloning of the cDNA for TFI and development of its entire proteinsequence and structural domains as disclosed herein permits detailedstructure-functional analyses and provides a foundation for study of itsbiosynthetic regulations. The invention thus is important to medicalscience in the study of the coagulation cascade with respect to agentswhich are able to inhibit Factor X_(a) and Factor VII_(a)/TF enzymaticcomplex.

DETAILED DESCRIPTION OF THE INVENTION

While the specification concludes with claims particularly pointing outand distinctly claiming the subject matter regarded as forming thepresent invention, it is believed that the invention will be betterunderstood from the following detailed description of preferredembodiments of the invention taken in conjunction with the appendeddrawings, in which:

FIG. 1 shows the screening of λgt11 clones with ¹²⁵I-Factor X_(a).Cloned phage lysates (0.1 ml) were spotted on a nitrocellulose paper bysuction using a dot blot apparatus. The nitrocellulose paper was thenprobed with ¹²⁵I-Factor X_(a) and autoradiographed as describedhereinafter. The clones that appear as dark spots are positive clonesthat bind ¹²⁵I-Factor X_(a). Control λgt11 (lower right corner) andother clones do not bind ¹²⁵I-Factor X_(a).

FIG. 2 shows a partial restriction map and sequencing strategy for theλP9 inserts. The scale at the bottom indicates the nucleotide position.The thick bar represents the coding region. The thin bars represent 5′-and 3′-noncoding regions. The restriction endonuclease sites wereconfirmed by digestion. The arrows show the overlapping M13 clones usedto sequence the cDNA.

FIG. 3 shows the nucleotide sequence and translated amino acid sequenceof the human TFI cDNA. Nucleotides are numbered on the left and aminoacids on the right. The underlined sequences have been independentlyconfirmed by amino acid sequence analysis of the purified TFI proteinand two V₈ protease+trypsin digested peptides. Amino acid+1 was assignedto the first methionine after a stop codon of the 5′-noncoding region.Potential N-lined glycosylation sites are marked by asterisks.

FIG. 4 is a graphical representation which shows the charge distributionof the amino acid sequence in TFI. Charges are calculated from the firstresidue to the i-th residues and displayed at the i-th residue. Thus thevalue of the i-th position is the summation of all charges from thefirst residue to the i-th residue and the difference of the chargesbetween the i-th and j-th residue (j>i) is the net charge of thefragment from i-th to j-th residue.

FIG. 5 is a graphical representation which shows the hydrophobicityprofile of TFI. The hydrophobicity profile was analyzed by a computerprogram whereby the hydrophobicity index of the amino acid residues isdefined as the depth to which an amino acid residue is buried inside aprotein (from X-ray crystallographic data) [Kidera et al., J. ProteinChem. 4, 23–55 (1985)]. The hydrophobicity profile along the sequencewas smoothed using the program ICSSCU in IMSL LIbrary [IMSL LibraryReference Manual, 9th ed., Institute for Mathematical and StatisticalSubroutine Library, Houston, Tex. (1982)].

FIG. 6 shows an alignment of the basic protease inhibitor domains of TFIwith other basic protease inhibitors. All the sequences except TFI wereobtained from the National Biomedical Research Foundation ProteinSequence Database (Georgetown University, Washington, D.C., release 13,June 1987). 1. Bovine basic protease inhibitor precursor; 2. Bovinecolostrum trypsin inhibitor; 3. Bovine serum basic protease inhibitor;4. Edible snail isoinhibitor K; 5. Red sea turtle basic proteaseinhibitor (only amino acids 1–79 presented); 6. Western sand viper venombasic protease inhibitor I; 7. Ringhals venom basic protease inhibitorII; 8. Cape cobra venom basic protease inhibitor II; 9. Russell's vipervenom basic protease inhibitor II; 10. Sand viper venom basic proteaseinhibitor III; 11. Eastern green mamba venom basic protease inhibitor Ihomolog; 12. Black mamba venom basic protease inhibitor B; 13. Blackmamba venom basic protease inhibitor E; 14. Black mamba venom basicprotease inhibitor I; 15. Black mamba venom basic protease inhibitor K;16. β-1-Bungarotoxin B chain (minor); 17. β-1-Bungarotoxin B chain(major); 18. β-2-Bungarotoxin B chain; 19. Horse inter-α-trypsininhibitor [amino acids 1–57(1); 58–123 (2)]; 20. Pig inter-α-trypsininhibitor [amino acids 1–57(1); 58–123(2)]; 21. Bovine inter-α-trypsininhibitor [amino acids 1–57(1); 58–123(2)]; 22. Humanα-1-microglobulin/inter-α-trypsin inhibitor precursor [amino acids227–283(1); 284–352(2)]; 23. TFI [amino-acids 47–117(1); 118–188(2);210–280(3)]. Gaps were included in 16, 17, 18 to achieve best alignment.Standard one letter codes for amino acids are used.

FIG. 7 shows the Northern blot analysis of RNAs from 3 liver-derivedcell lines. Ten μg of poly(A)⁺ RNA were used per lane. Lane 1, Changliver cell; lane 2, SK-HEP-1 hepatoma cell; lane 3, HepG2 hepatoma cell.

Standard biochemical nomenclature is used herein in which the nucleotidebases are designated as adenine (A); thymine (T); guanine (G); andcytosine (C). Corresponding nucleotides are, for example,deoxyguanosine-5′-triphosphate (dGTP). As is conventional forconvenience in the structural representation of a DNA nucleotidesequence, only one strand is shown in which A on one strand connotes Ton its complement and G connotes C. Amino acids are shown either bythree letter or one letter abbreviations as follows:

Abbreviated Designation Amino Acid A Ala Alanine C Cys Cysteine D AspAspartic acid E Glu Glutamic acid F Phe Phenylalanine G Gly Glycine HHis Histidine I Ile Isoleucine K Lys Lysine L Leu Leucine M MetMethionine N Asn Asparagine P Pro Proline Q Gln Glutamine R Arg ArginineS Ser Serine T Thr Threonine V Val Valine W Trp Tryptophan Y TyrTyrosine

Commonly available restriction endonucleases described herein have thefollowing restriction sequences and (indicated by arrows) cleavagepatterns:

EcoRl  ↓ GAATTC CTTAAG      ↑ Ssp1    ↓ AATATT TTATAA    ↑ Cla1   ↓ATCGAT TAGCTA     ↑ Alu1   ↓ AGCT TCGA   ↑ Stu1    ↓ AGGCCT TCCGGA    ↑

In order to illustrate specific preferred embodiments of the inventionin greater detail, the following exemplary laboratory preparative workwas carried out.

EXAMPLE 1

Materials

Human placental and fetal liver cDNA libraries were obtained fromClonetech. The protoblot immunoscreening kit was purchased from PromegaBiotech. Restriction enzymes were from New England Biolabs. Calfintestine alkaline phosphatase, T4 DNA ligase, DNA polymerase I(Klenow), exo-nuclease III and S1 nuclease were from BoehringerMannheim. dNTPs were from P. L. Biochemicals. 5′-[α-³⁵S]-thio-dATP (600Ci/mmol) was from Amersham. Sequencing kit (Sequenase) was from UnitedStates Biochemicals. Chang liver cells (ATCC CCL 13) and HepG2 hepatomacells (ATCC HB 8065) were obtained from the American Type CultureCollection. SK-HEP-1 hepatoma cells were originally derived from a liveradenocarcinoma by G. Trempe of Sloan-Kettering Institute for CancerResearch in 1971 and are now widely and readily available.

¹²⁵I-Factor X_(a) was prepared by radio-labeling using Iodo-gen. Thespecific activity was 2000 dpm/ng. Greater than 97% of radioactivity wasprecipitable with 10% trichloroacetic acid (TCA). The iodinated proteinretained >80% of their catalytic activity toward Spectrozyme X_(a)(American Diagnostica product).

An anti-TFI-Ig Sepharose® 4B column was prepared as follows: A peptide(called TFI-peptide) containing a sequence corresponding to the aminoacid sequence 3–25 of the mature TFI was synthesized using Biosystem'ssolid phase peptide synthesis system. The TFI-peptide (5 mg) wasconjugated to 10 mg of Keyhole lympet hemocyanin by glutaraldehyde. TwoNew Zealand white rabbits were each immunized by intradermal injectionwith a homogenate containing 1 ml of Freund complete adjuvant and 1 mlof conjugate (200 μg of TFI-peptide). One month later the rabbits wereeach boosted with a homogenate containing 1 ml of Freund incompleteadjuvant and 1 ml of conjugate (100 μg of conjugate). Antiserum wascollected each week for 3 months and booster injections were performedmonthly. To isolate specific antibody against TFI-peptide, the antiserumwas chromatographed on a TFI-peptide Sepharose 4B column. The column waswashed with 10 volumes of PBS (0.4 M NaCl-0.1 M benzamidine-1% Triton®X-100) and the same solution without Triton X-100. The antibody waseluted with 0.1 M glycine/HCl, pH 2.2, immediately neutralized by adding1/10 volume of 1 M Tris-OH and dialyzed against saline solution. Theisolated antibody was coupled to cyanogen bromide activated Sepharose 4Bby the manufacturer's (Pharmacia) method and used to isolate TFI fromthe cell culture medium.

Chang liver cell was cultured by the method described previously byBroze and Miletich, Proc. Natl. Acad. Sci. USA 84, 1886–1890 (1987). Theconditioned medium was chromatographed on the anti-TFI-Ig Sepharose 4Bcolumn. The column was washed with 10 volumes of PBS-1% Triton X-100 andPBS. The bound TFI was eluted with 0.1 M glycine/HCl, pH 2.2. Theimmunoaffinity isolated TFI was further purified by preparative sodiumdodecylsulfate polyacrylamide gel electrophoresis (Savant apparatus).Amino acid analysis of the final product showed the same amino terminalsequence as the TFI isolated from HepG2 cells as described in copendingapplication, Ser. No. 77,366, filed Jul. 23, 1987. The isolated Changliver TFI was then used to immunize rabbits by the immunization protocoldescribed above. The antiserum obtained had a titer of about 100 μg/mlin the double immunodiffusion test. This antiserum was used in theimmuno-screening of λgt11 cDNA libraries.

Methods

Isolation of cDNA clones. Methods for screening the placental and fetalliver cDNA libraries with antibody, plaque purification, and preparationof λ-phage lysate and DNA were as described by Wun and Kretzmer, FEBSLett. 1, 11–16 (1987). The antiserum was pre-adsorbed with BNN97 λgt11lysate and diluted 1/500 for screening the library.

Screening of Factor X_(a) Binding Activity

Recombinant proteins induced by isopropyl-β-thiogalactoside fromimmuno-positive λ-phage isolates or from control λgt11 were screened forFactor X_(a) binding activity. The λ-phage lysates (0.1 ml) werefiltered through a nitrocellulose paper using a dot-blot apparatus (BioRad). The nitrocellulose paper was then immersed and agitated in aphosphate buffered saline containing 5 mg/ml bovine serum albumin and2.5 mg/ml bovine gamma globulin at room temperature for 1 h. Thesolution was replaced with ¹²⁵I-Factor X_(a) (1.0×10⁶ cmp/ml) dissolvedin the same solution supplemented with 0.1 mg/ml heparin and theagitation continued for another hour. The nitrocellulose paper was thenwashed with phosphate buffered saline containing 0.05% Tween® 20. Thewashing buffer was changed every 5 min., 4 times. The nitrocellulosepaper was then air-dryed and prepared for autoradiography using KodakXR5 film. The film was developed after 1 week exposure.

Preparation of poly(A)⁺ RNA and Northern blotting. Total RNAs wereprepared from cultured Chang liver cell, HepG2 hepatoma cell andSK-HEP-1 hepatoma cell using the sodium perchlorate extraction method ofLizardi, and Engelberg, Anal. Biochem. 98, 116–122, (1979). Poly(A)⁺RNAs were isolated by batch-wise adsorption on oligo(dT)-cellulose (P-LBiochemical, type 77F) using the procedure recommended by themanufacturer. For Northern blot analysis, 10 μg each of poly(A)⁺ RNA wastreated with glyoxal [Thomas, Methods Enzymol. 100, 255–266 (1983)] andsubjected to agarose gel electrophoresis in a buffer containing 10 mMsodium phosphate, pH 7.0. Bethesda Research Laboratory's RNA ladder wasused as a molecular weight marker. The RNAs were transblotted onto anitrocellulose paper which was then baked at 80° for 2 h. The insert DNAof λP9 clone was radiolabeled with ³²P by nick translation and used as aprobe [Maniatis et al., Molecular Cloning: A Laboratory Model, ColdSpring Laboratory, Cold Spring Harbor, N.Y., (1982)]. The blot washybridized with 5×10⁶ cpm of the probe in 5 ml of a solution containing50% formamide, 5×SSC, 50 mM sodium phosphate, pH 7.0, 250 μg/mldenatured salmon sperm DNA, and 1× Denhardt's solution at 42° for 16 h.The filter was washed in 0.1% sodium dodecylsulfate (SDS), 2×SSC at roomtemperature 3 times, each time 5 min., and in 0.1% SDS, 0.2×SSC at 50°twice, each 5 min. The nitrocellulose paper was then air dried,autoradiographed for 3 days at −70° using Kodak XAR-5 film andintensifying screen.

Other recombinant DNA methods. Preparation of cloned λgt11 DNA,subcloning in pUC19 plasmid and M13 mp18 vector, generation of deletionby exonuclease III digestion and DNA sequencing by dideoxy method[Sanger et al., Proc. Natl. Acad. Sci. USA 83, 6776–6780 (1977)], wereperformed as described by Wun and Kretzmer, supra.

The program FASTP written by Lipman and Pearson, Science 227 1435–1441(1985), was used to identify homologous families of proteins fromNational Biomedical Research Foundation Sequence Data Bank (release 13,June 1987) and to align the sequences within the homologous family.

RESULTS

Screening of cDNA Libraries

A number of cell lines were screened for the presence of TFI in theconditioned media and it was found that several liver-derived celllines, Chang liver, HepG2 hepatoma, and SK-HEP-1 hepatoma secrete TFI inculture. Initially, an antiserum against TFI was used to screen a humanfetal liver λgt11 cDNA library (10⁶ plaque forming units), and 15immunologically positive clones were obtained. Subsequently, the samemethod was used to screen a placental λgt11 cDNA library. Out of 10⁶plaque forming units, 10 immunologically positive clones were obtained.These clones were plaque purified and the lysates of the purified cloneswere tested for the functional activity of TFI. Theisopropylthio-galactoside induced phage lysates were absorbed on thenitrocellulose paper and screened for the ¹²⁵I-Factor X_(a) bindingactivity. FIG. 1 demonstrates that some of these immunologicallypositive clones showed the ability to bind the ¹²⁵I-Factor X_(a) on thenitrocellulose paper. In all, 3 out of 15 immunologically positive fetalliver clones, and 4 out of 10 immunologically positive placental clonesshowed ¹²⁵I-Factor X_(a) binding activity. These immunologically andfunctionally positive clones were digested with EcOR1 and the size ofthe inserts were estimated by gel electrophoresis. One clone fromplacental library (λP9) had an insert of approximately 1.4 kb, while allthe other clones contain inserts of approximately 1.0 kb. Partial DNAsequencing has shown that 1.0 kb clones contain sequences identical topart of the longer 1.4 kb placental clone (λP9). The λP9 was thereforeselected for complete sequencing.

Nucleotide Sequence and Predicted Protein Sequence of TFI cDNA Isolate

The λP9 clone was subjected to restriction mapping, M13 subcloning andsequencing by the strategy shown in FIG. 2. The entire sequence wasdetermined on both strands by the exonuclease III deletion method[Henikoff, Gene 28, 351–359 (1984)] and found to consist of 1432 basesin length. The sequence is shown in FIG. 3. It contains a 5′-noncodingregion of 133 bases, an open reading frame of 912 nucleotides, and a3′-noncoding region of 387 nucleotides. The first ATG occurs atnucleotide 134 in the sequence TAGATGA which was closely followed by asecond ATG at nucleotide 146 in the sequence ACAATGA. These are possiblythe initiation sequences, although they differ from the proposedconsensus sequence for initiation by eukaryotic ribosome, ACCATGG[Kozak, Cell 44, 283–292. (1986)]. Twenty-eight amino acids precede asequence corresponding to the N-terminal of the mature protein. Thelength and composition of the hydrophobic segment of these 28 aminoacids are typical of signal sequences [Von Heijne, Eur. J. Biochem. 133,17–21 (1983); J. Mol. Biol. 184, 99–105 (1985)]. A signal peptidasepossibly cleaves at Ala₂₈-Asp₂₉ to give rise to a mature protein. Thesequence predicted for mature TFI consists of 276 amino acids thatcontains 18 cysteine residues and 7 methionines. The calculated mass of31,950 Daltons based on the deduced protein sequence for mature TFI issomewhat lower than the 37–40 kDa estimated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis of isolated protein, and thedifference probably reflects the contribution of glycosylation to themobility of the natural protein. The deduced protein sequencecorresponding to the mature protein contains 3 potential N-linkedglycosylation sites with the sequence Asn-X-Thr/Ser (amino acidpositions 145, 195, and 256). Amino acid sequence analysis of purifiedwhole TFI and two isolated proteolytic fragments match exactly theprotein sequence deduced from cDNA sequence (FIG. 3, underlined),indicating the isolated cDNA clone encodes TFI. The 3′-noncoding regionis A+T rich (70% A+T). Neither consensus polyadenylation signal, AATAAA[Proudfoot and Brownlee, Nature 252, 359–362 (1981)] nor the poly A tailwas found in this clone, possibly due to artefactual loss of part of 3′terminal portion during construction of the library.

Charge Distribution, Hydrophobicity/Hydrophilicity, and InternalHomology

The translated amino acid sequence of the TFI contains 27 lysines, 17arginines, 11 aspartic acids, and 25 glutamic acids. The chargedistribution along the protein is highly uneven as shown in FIG. 4. Thesignal peptide region contains 2 positively charged lysine with 26neutral residues. The amino-terminal region of the mature proteincontains a highly negatively charged stretch. Six of the first 7residues are either aspartic acid or glutamic acid which are followedclosely by two more negatively charged amino acids downstream before apositively charged lysine residue appears. The center portion of themolecule is generally negatively charged. At the carboxy terminal, thereis a highly positively charged segment. The amino acids 265 to 293 ofTFI contain 14 positively charged amino acids including a 6-consecutivearginine+lysine residues.

The predicted hydrophilicity/hydrophobicity profile of TFI protein isshown in FIG. 5. The signal peptide contains a highly hydrophobic regionas expected. The rest of the molecule appears rather hydrophilic.

The translated amino acid sequence of TFI contains several discernibledomains. Besides the highly negatively charged N-terminal domain and thehighly positively charged C-terminal domain, the center portion consistsof 3 homologous domains which have the typical sequences of theKunitz-type inhibitors (see below).

Homology to Other Proteins

By searching the National Biomedical Research Foundation sequence database, it was found that the N-terminal domain and C-terminal domain ofTFI do not show significant homology to other known proteins. The 3internal homologous domains, however, are each homologous to thesequences of other basic protease inhibitors including bovine pancreaticbasic protease inhibitor (aprotinin), venom basic protease inhibitors,and inter-α-trypsin inhibitors (FIG. 6). It is noteworthy that disulfidebonding structure is highly conserved in all these inhibitors. Based onthese homologies, it is clear that TFI belongs to the basic proteaseinhibitor gene superfamily.

Northern Blotting

Poly(A)+ RNAs were purified from TFI-producing liver-derived cell lines,Chang liver, HepG2 hepatoma, and SK-HEP-1 hepatoma cells. The poly (A)+RNAs were resolved by denaturing agarose gel electrophoresis,transblotted onto a nitrocellulose paper and probed with ³²P-labeled TFIcDNA (λP9). As shown in FIG. 7, two major bands of hybridization wereobserved that corresponded to mRNAs of 1.4 kb and 4.4 kb in all threecell lines tested. Several other cell lines were tested which do notproduce detectable amounts of TFI and in which no hybridization with theprobe was found. (data not shown).

Various other examples will be apparent to the person skilled in the artafter reading the present disclosure without departing from the spiritand scope of the invention. It is intended that all such furtherexamples be included within the scope of the appended claims.

1. A method for isolating tissue factor inhibitor (TFI) comprising aminoacids 29 to 304 of SEQ ID NO:2 comprising: culturing a liver cell linein conditioned medium; and isolating TFI from the medium.
 2. The methodof claim 1 wherein the TFI is isolated from the medium bychromatography.
 3. The method of claim 2 wherein chromatography isperformed using a TFI antibody coupled to a resin.
 4. The method ofclaim 3 wherein the resin is Sepharose 4B.
 5. The method of claim 1wherein the liver cell line is a Chang liver cell line.
 6. The method ofclaim 1 wherein the liver cell line is a HepG2 hepatoma cell line. 7.The method of claim 1 wherein the liver cell line is a SK-HEP-1 hepatomacell line.